Heat-inducible self-assembling protein domains

ABSTRACT

A heat-inducible self-assembling fusion protein that includes a self-assembly domain and a target protein, wherein the self-assembly domain remains folded during assembly. The aggregate forming fusion protein can be induced to form protein aggregates conjugated to a target protein. The aggregates can be used similarly to beads in many laboratory protocols and other applications. Also disclosed are methods of making and using the protein aggregates.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority of U.S. ProvisionalPatent Application No. 62/068,390, filed Oct. 24, 2014, which is herebyincorporated by reference in its entirety.

BACKGROUND OF THE INVENTION A. Field of the Invention

The invention generally concerns molecular biology and proteinengineering. In particular, it involves proteins that are able toself-assemble in a heat-inducible fashion.

B. Description of Related Art

In many scientific applications, conjugation of molecules to largeralbeit still microscopic particles (e.g., spheres, beads, rods,nanoparticles) is used to immobilize, control, partition, or otherwisemanipulate other molecules. Sigma-Aldrich, Thermo Fisher Scientific, andmany other companies sell particles made of sepharose, agarose, or otherpolymers which either come pre-conjugated with molecules (for example,antibodies, peptides, protein A, streptavidin, biotin) or can beconjugated by the customer using crosslinking chemistry. These particlescan also be magnetic, allowing their rapid removal with a magnet.

Genetically encodable, heat-inducible, particle-forming protein domainwould be valuable, as it would allow genetic engineering techniques thatare now commonplace to be used to construct conjugated particles.

SUMMARY OF THE INVENTION

The present application provides heat-inducible, self-assembling proteindomains and fusion proteins including such domains that can be used inmany different applications described herein. For example, fusionproteins incorporating a self-assembly domain and a target proteinprovide improved, rapid purification methods. Such fusion proteins canbe genetically encoded, expressed, and purified using conventionallaboratory techniques. The fusion proteins can form protein aggregatesupon heat induction, and can be used in the place of beads or otherconjugated particles in many laboratory protocols.

Proteins containing a self-assembly domain form insoluble aggregatesrapidly upon heat treatment, but are essentially absent and soluble attemperatures at or below 30° C., including room temperature. Aself-assembly domain, fused to other proteins, confers self-assemblingability on these proteins. Folded proteins retain function within theassembled protein aggregates, and RNA, DNA, and small molecules can bestably bound within the assembled protein aggregates.

Disclosed herein is a self-assembling fusion protein comprising: (a) aheat-inducible self-assembly domain; and (b) a target protein; whereinthe self-assembly domain remains folded (at least partially or mostly)during assembly. In some embodiments, the fusion protein is capable ofself-assembling into protein aggregates by being heated to a temperatureof between about 35 and 50° C. or any range derivable therein. In someembodiments, the heat induction is at a temperature greater than, lessthan, or between any two of about 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, and 55° C.

In some embodiments of the disclosure, the fusion protein formsaggregates in less than or exactly 1200, 1100, 1000, 900, 800, 700, 600,500, 400, 300, 275, 250, 225, 200, 175, 150, 125, 100, 75, 60, or 30seconds upon heat induction, or any derivable range therein.

In some embodiments, the self-assembly domain is a GST-like domain or apolypeptide with at least 90% identity to a GST-like domain. A GST-likedomain refers to a conserved protein domain known in the art as aGlutathione S-transferase C-terminal-like domain. This conserved domainis described in the NCBI database of conserved domains (See alsoMarchler-Bauer A. et al. (2013), “CDD: conserved domains and proteinthree-dimensional structure.” Nucleic Acids Res. 41(D1):D384-52), whichis hereby incorporated by reference. Furthermore, a protein can bedetermined to have this conserved domain by inputting the proteinsequence into the NCBI conserved domain database, which can be found onthe world wide web at ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi. Thedomain is capable of making stable protein-protein interactions betweenitself and another GST-like domain, in one of two orientations, as shownin Simader H et al. (2006), “Structural basis of yeast aminoacyl-tRNAsynthetase complex formation revealed by crystal structures of twobinary sub-complexes,” Nucleic Acids Res. 34(14):3968-79.

The term “identity,” “homology” or “similarity” refers to sequencesimilarity between two peptides or between two nucleic acid molecules. Adegree of identity can be determined by comparing a position in eachsequence which may be aligned for purposes of comparison. When aposition in the compared sequence is occupied by the same base or aminoacid, then the molecules are homologous at that position. A degree ofidentity between sequences is a function of the number of matchingpositions shared by the sequences. An “unrelated” or “non-homologous”sequence shares less than 40% identity, though preferably less than 25%identity, with one of the sequences of the present invention. Thepercent identity can be calculated by the formula: (Matches×100)/Lengthof aligned region (with gaps). Note that only internal gaps are includedin the length, and not gaps at the sequence ends.

A polynucleotide or polynucleotide region (or a polypeptide orpolypeptide region) having a certain percentage (for example, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, 98% or 99%, or any range derivabletherein) of “sequence identity” or “homology” to another sequence meansthat, when aligned, that percentage of bases (or amino acids) are thesame in comparing the two sequences. This alignment and the percenthomology or sequence identity can be determined using software programsknown in the art, for example those described in Ausubel et al. eds.(2007) Current Protocols in Molecular Biology.

In some embodiments, a polypeptide may have at least 65, 70, 75, 80, 85,90, 95, 96, 97, 98, 99, or 100% identity (or any range derivablethereof) with another polypeptide.

In some embodiments, the self-assembly domain comprises a polypeptidefrom Arc1, Mes1, Gus1, or a polypeptide with at least 90% identity toArc1, Mes1, or Gus1. Arc1, Mes1, and Gust are proteins with GST-likedomains. In some embodiments, the self-assembly domain comprises apolypeptide that is at least 20 amino acids in length and has at least90% identity to the first 250 amino acids to Arc1, Mes1, or Gus1. Inthis case, the percent identity is calculated specifically as describedabove, wherein the sequence is aligned, and internal gaps are used tocalculate sequence identity, but gaps at the end of the alignment arenot used in calculating sequence identity. In some embodiments, theself-assembly domain comprises a polypeptide that is at least 20, 30,40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180,190, or 200 amino acids in length and has at least 90% identity to apolypeptide of similar or the same length from the first 250 amino acidsto Arc1, Mes1, or Gus1. In some embodiments, the self-assembly domaincomprises a polypeptide that is at least 10, 20, 30, 40, 50, 60, 70, 80,90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 amino acidsin length, or any derivable range thereof. The polypeptide may have acertain degree of identity to the first (N-terminus) 50, 75, 100, 125,150, 175, 191, 200, 225, 250, 300, or 300 amino acids (or any derivablerange thereof) of Arc1, Mes1, or Gus1. In some embodiments, theself-assembly domain comprises a polypeptide from Gus1 or a polypeptidewith at least 90, 95, 97, or 99% identity (or any range derivablethereof) to Gus1.

In some embodiments, the self-assembly domain comprises a polypeptidefrom Tef3, Tef4, Efb1, or a polypeptide with at least 90% identity toTef3, Tef4, or Efb1. Tef3, Tef4, and Efb1 are proteins with GST-likedomains. In some embodiments, the self-assembly domain comprises apolypeptide that is at least 20 amino acids in length and has at least90% identity to 20 amino acids of the first 250 amino acids from Tef3,Tef4, or Efb1. In this case, the percent identity is calculatedspecifically as described above, wherein the sequence is aligned, andinternal gaps are used to calculate sequence identity, but gaps at theend of the alignment are not used in calculating sequence identity. Insome embodiments, the self-assembly domain comprises a polypeptide thatis at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140,150, 160, 170, 180, 190, or 200 amino acids in length (or any rangederivable therein) and has at least 90% identity to a polypeptide ofsimilar or the same length from the first 250 amino acids of Tef3, Tef4,or Efb1. In some embodiments, the self-assembly domain comprises apolypeptide that is at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100,110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 amino acids inlength, or any derivable range thereof. The polypeptide may have acertain degree of identity to the first (N-terminus) 50, 75, 100, 125,150, 175, 191, 200, 225, 250, 300, or 300 amino acids (or any derivablerange thereof) of Tef3, Tef4, or Efb1.

In some embodiments, the self-assembly domain comprises a polypeptidefrom Yef3, Ura7, or a polypeptide with at least 90% identity to Yef3 orUra7.

In some embodiments, the polypeptide is from a Saccharomyces cerevisiaeprotein. In some embodiments, the polypeptide of the assembly domain isfrom a homolog of Arc1, Mes1, Gus1, Tef3, Tef4, Efb1, Yef3, or Ura7 fromanother organism. For example, the polypeptide may be a homolog of Arc1,Mes1, Gus1, Tef3, Tef4, Efb1, Yef3, or Ura7 from B. dendrobatidis, U.hordei, U. maydis, S. reilianum, P. triticina, P. graminis, W. sebi, M.globosa, M. larici-populi, F. radiculosa, P. indica, S. lacrymans, C.cinerea, P. carnosa, A. bisporus, Allavus, C. neoformans, C. gattii, A.oligospora, Lelongisporus, N. tetrasperma, C. parapsilosis, C. albicans,D. hansenii, C. dubliniensis, C. lusitaniae, P. sorbitophila, R.delemar, T. melanosporum, P. pastoris, S. stipitis, S. japonicus, C.tenuis, W. ciferrii, P. angusta, T. stipitatus, S. pombe, A. terreus, N.fumigata, P. marneffei, N. fischeri, A. clavatus, A. oryzae, P.digitatum, P. chrysogenum, A. niger, A. kawachii, T. verrucosum, N.dairenensis, P. brasiliensis, A. benhamiae, A. capsulata, A. gypseum, K.lactis, N. castellii, A. dermatitidis, T. rubrum, T. equinum, S.arboricola, S. cerevisiae, T. blattae, K. naganishii, C. posadasii, A.gossypii, V. polyspora, T. phaffii, A. otae, Z. rouxii, T. delbrueckii,K. africana, C. glabrata, L. thermotoleran, Y. lipolytica, E. aedis, C.militaris, U. reesii, P. firoveci, E. nidulans, T. tonsurans, T. asahii,S. commune, L. maculans, P. teres, N. sp., N. parisii, E. hellem, E.cuniculi, E. romaleae, E. intestinalis, P. tritici-repen, C. globosum,T. terrestris, G. clavigera, T. heterothallic, B. bassiana, M.phaseolina, V. corneae, C. thermophilum, N. crassa, M. oryzae, G.graminis, H. atroviridis, H. vixens, Hjecorina, V. culicis, T. hominis,S. sclerotiorum, B. fuckeliana, N. haematococca, M. robertsii, E.bieneusi, M. acridum, F. oxysporum, C. graminicola, C. gloeosporioid, G.destructans, G. lozoyensis, C. higginsianum, M. brunnea, V. dahliae, F.pseudogramine, S. macrospora, N. ceranae, V. albo-atrum, S.passalidarum, C. tropicalis, M. guilliermond, or E. dermatitidis.

The systematic name and common name of proteins that are useful for theself-assembly domain are:

Systematic Common Name Name YGL105W Arc1 YGR264C Mes1 YGL245W Gus1YPL048W Tef3 YKL081W Tef4 YAL003W Efb1 YLR249W Yef3 YBL039C Ura7

In some embodiments, the self-assembly domain comprises a polypeptide ofSEQ ID NO:9, 10, 11, or 12, a fragment thereof, or a polypeptide with atleast 90% identity to SEQ ID NO:9, 10, 11, or 12, or a fragment thereof.SEQ ID NOs:9-12 represents the N-terminal GST-like domains of Gus1,Mes1, Tef4, and Tef3.

In some embodiments, the majority of the fusion protein moleculesaggregate to form direct protein-protein interactions with other fusionprotein molecules upon heat-induction. “Direct” protein-proteininteractions are not mediated by solvent or any other molecule, butinvolve the direct non-covalent interaction of amino acids with otheramino acids. Theses direct interactions may be aromatic-aromatic,cation-aromatic, electrostatic, van der Walls, or hydrophobicinteractions. The fusion protein is capable of forming the proteinaggregates under relatively “mild” conditions, which is one way that theprotein aggregates described herein are more useful than other types ofprotein aggregates for laboratory applications such as formingaggregates conjugated to target proteins, which aggregates can be usedin the place of beads or solid substrates in many laboratory protocols.The term “aggregate” in the prior art may refer to mis-folded proteinsunder denaturing conditions (e.g., elevated temperatures, pH, high saltcontent). However, the term “protein aggregates,” as used herein, is notmeant to refer to mis-folded protein, but instead refers to protein thatsubstantially retains a tertiary structure, but associates with theself-assembly domain of any other proteins in the composition. Theself-assembly domain, while aggregated, substantially retains functionand a tertiary structure. While the self-assembly domain does undergo aconformational change after a temperature shift, the self-assemblydomain is not mis-folded at the temperature shift and substantiallyretains a tertiary structure. Therefore, in some embodiments, theself-assembly domain remains folded at a temperature below, above, orany range derivable thereof, of 60, 55, 50, 45, 40, 35, 30, 25, or 20°C. In some embodiments, the self-assembly domain remains folded at atemperature range of 20−50° C.

In some embodiments, the self-assembly domain is not an elastin-likepolymer (ELP), does not have a significant degree of homology to an ELP,and/or does not comprise an ELP polypeptide or fragment.

In some embodiments, the fusion proteins do not self-assemble attemperatures below about 35° C. In some embodiments, the fusion proteinsdo not self-assemble at temperatures below about 40, 35, 30, 25, 20, or15° C. (or any range derivable therein).

In some embodiments, the self-assembly domain is at least, at most, orexactly 15, 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 300, 350,400, 500, 600, 700, 800, 900, and 1000 amino acids in length, or anyrange derivable therein.

In some embodiments, the target protein or polypeptide is at least, atmost, or exactly 5, 10, 15, 25, 50, 75, 100, 125, 150, 175, 200, 225,250, 300, 350, 400, 500, 600, 700, 800, 900, and 1000 amino acids inlength, or any range derivable therein.

The target protein may be a protein in which purification is desired ormay be a component of an assay, such as an antibody or protease. In someembodiments, the target protein is ferritin, a fluorescent protein, anantibody, an antibody fragment, protein A, streptavidin, protein G,protein A/G, protein L, a protease, or StrepTactin. In some embodiments,the target protein is a protease. In some embodiments, the targetprotein is a wild-type or mutant protein from a eukaryote or aprokaryote. In some embodiments, the target protein is not a naturallyoccurring protein.

In some embodiments, the fusion protein further comprises a proteasecleavage site. In some embodiments, the protease cleavage site isbetween the target protein and the self-assembly domain.

Further aspects of the disclosure relate to a protein aggregatecomprising the fusion protein described herein. In some embodiments, theprotein aggregate further comprises a nucleic acid or protein that isspecifically bound to the target protein. In some embodiments, a smallmolecule is specifically bound to the target protein. Further aspectsrelate to an aqueous composition comprising the fusion protein or theprotein aggregate described herein.

In some embodiments, the protein aggregate is in an aqueous composition,wherein the aqueous composition comprises between about 0 and 500 mM KClor NaCl, or is about or is less than any one of or between any two ofabout 50, 100, 150, 200, 250, 300, 350, 400, 450, and 500 mM KCl orNaCl. In some embodiments, the aqueous composition does not includealcohol. In some embodiments the fusion protein is present in theaqueous composition at a concentration of between about 5 and 50 μM, orat a concentration of less than any one of or between any two of about5, 10, 15, 20, 25, 30, 35, 40, 45, and 50 μM. In some embodiments, thefusion protein is at least 55% pure, or is at least between any two ofabout 55, 60, 65, 70, 75, 80, 85, 90, 95, 98, 99, and 99.9% pure in theaqueous solution. In some embodiments, the pH of the aqueous compositionis between about 6.0 and 8.0, or is about or is greater than, less than,or between any two of about 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8,6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, and 8.0.

In some embodiments, the target protein in the fusion proteins describedabove is a restriction enzyme, DNA polymerase, protease, ligase, RNApolymerase, methylase, polyadenylate polymerase, topoisomerase, guanylyltransferase, ribonuclease, deoxyribonuclease, alkaline phosphatase,polynucleotide kinase or reverse transcriptase. In some embodiments, thetarget protein is a therapeutic protein.

Further aspects of the disclosure relate to a polynucleotide coding forthe fusion protein described herein. Other embodiments of the disclosurerelate to a host cell comprising the fusion protein or thepolynucleotide described herein. Yet further aspects relate to celllysate comprising the fusion protein as described herein.

Also disclosed is a method for aggregating a target protein comprising:formulating an aqueous composition comprising the fusion protein asdescribed herein; and heating the composition to a temperature betweenabout 35 and 50° C. or any range derivable therein. In some embodiments,the temperature is above, below, or a derivable range of about 20, 25,30, 35, 40, 45, 50, 55, or 60° C.

Further method aspects of the disclosure relate to a method ofselectively depleting a molecule from an aqueous composition comprising:formulating an aqueous composition comprising the molecule and thefusion protein as described herein, wherein the target protein is aprotein that specifically binds to the molecule; heating the aqueouscomposition to a temperature between about 35 and 50° C. to form proteinaggregates comprising the fusion protein and the molecule; and removingthe protein aggregates from the aqueous composition. In someembodiments, the temperature is above, below, or a derivable range ofabout 20, 25, 30, 35, 40, 45, 50, 55, or 60° C. In some embodiments, themolecule is a nucleic acid. The molecule may be, for example, a DNA or aRNA. In some embodiments, removing the protein aggregates from theaqueous composition is performed by centrifuging or filtering theaqueous composition.

Further method aspects of the disclosure relate to a method ofselectively depleting a molecule from an aqueous composition comprising:heating the aqueous composition comprising the fusion protein to atemperature between about 35 and 50° C. to form protein aggregatescomprising the fusion protein; adding the molecule to the aqueouscomposition comprising the aggregated fusion protein, wherein the targetprotein is a protein that specifically binds to the molecule; andremoving the protein aggregates from the aqueous composition. In someembodiments, the temperature is above, below, or a derivable range ofabout 20, 25, 30, 35, 40, 45, 50, 55, or 60° C. In some embodiments, themolecule is a nucleic acid. The molecule may be, for example, a DNA or aRNA. In some embodiments, removing the protein aggregates from theaqueous composition is performed by centrifuging or filtering theaqueous composition.

The removal or separation of the protein aggregates may be done bymethods known in the art for separating soluble and insoluble (proteinaggregates) fractions. These include, for example, centrifugation,filtration, and size exclusion chromatography.

Other aspects relate to a method of immunoprecipitating a moleculecomprising: formulating an aqueous composition comprising the moleculeand the fusion protein of any one of claims 1-20; wherein the targetprotein is an antibody or antigen binding fragment that specificallybinds to the molecule; and heating the aqueous composition to atemperature between about 35 and 50° C. to form protein aggregatescomprising the fusion protein and the molecule. In some embodiments, thetemperature is above, below, or a derivable range of 20, 25, 30, 35, 40,45, 50, 55, or 60° C. In some embodiments, the method further comprisesdetecting the molecule bound in the protein aggregate. In someembodiments, the method further comprises quantifying the molecule boundin the protein aggregate. In some embodiments, the method furthercomprises separating the protein aggregates from the solublecomposition.

Further method aspects relate to a method for purifying a proteincomprising: formulating an aqueous composition comprising a fusionprotein as described herein; heating the aqueous composition to atemperature between about 35 and 50° C. to form protein aggregatescomprising the fusion protein; and separating the protein aggregatesfrom the aqueous composition. In some embodiments, the temperature isabove, below, or a derivable range of 20, 25, 30, 35, 40, 45, 50, 55, or60° C. In some embodiments, the fusion protein is a first fusion proteincomprising a protein cleavage site between the self-assembly domain andthe target protein. In some embodiments, the method further comprises:cleaving the first fusion protein by formulating an aqueous compositioncomprising a second fusion protein and the first fusion protein in theseparated aggregate; wherein the target protein of the second fusionprotein is a protease that cleaves the first fusion protein at theprotein cleavage site between the self-assembly domain and the targetprotein of the first fusion protein; heating the aqueous composition toa temperature between about 35 and 50° C. to form protein aggregatescomprising the second fusion protein and the self-assembly domain of thecleaved first fusion protein; wherein the target protein of the firstfusion protein remains soluble; and separating the protein aggregatesfrom the soluble target protein of the first fusion protein. Thispurification method is further described in the figures and examples.

Further aspects relate to a method of immunoprecipitating or purifying amolecule comprising the steps of: formulating a first compositioncomprising the fusion protein as described herein; wherein the targetprotein is a first target protein that specifically binds to themolecule; heating the first composition to a temperature between about35 and 50° C. to form protein aggregates comprising the fusion protein;and contacting the first composition with a second compositioncomprising the molecule. In some embodiments, the first target proteinthat specifically binds to the molecule is an antibody, an antigenbinding fragment, or an affinity tag (e.g., PDZ domain). In someembodiments, the composition is heated to at least, at most, or exactlyabout 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 65, or 70°C., or any derivable range therein. In some embodiments, the heating ofthe composition is done prior to contacting of the first compositionwith the second composition. In some embodiments, the heating of thefirst composition is done after contacting the first composition withthe second composition.

In some embodiments, the second composition maintains a temperature ofless than 40° C. throughout the method. In some embodiments, the secondcomposition and/or second target protein maintains a temperature of lessthan 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29,28, 27, 26, 25, 24, 23, 22, 21, or 20° C. throughout the method. In someembodiments, contacting the first composition with the secondcomposition comprises mixing the compositions. The mixing may be done bymechanical means such as, for example, vortexing, pipetting, etc. Insome embodiments, the molecule is a fusion protein between a secondtarget protein and a tag that binds to the first target protein. In someembodiments, the first target protein is a PDZ domain. In someembodiments, the tag is a Ctag. In some embodiments, the moleculecomprises a protease cleavage site between the second target protein andthe tag. In some embodiments, the protease cleavage site is one known inthe art or described herein. In some embodiments, the method furthercomprises purifying the molecule by separating the aggregated proteinfrom the first and second composition. The separating may be done bymethods known in the art such as pelleting the aggregated proteins (i.e.centrifugation) or other separation techniques based on size and charge,for example. In some embodiments, the method further comprises elutingthe second target protein. In some embodiments the elution is done byadding an eluting peptide or peptide that competes for the binding tothe first target protein. In some embodiments, the eluting peptide is apeptide of SEQ ID NO:14 or a peptide having at least 90% sequenceidentity to SEQ ID NO:14. In some embodiments, the method furthercomprises contacting the molecule with a protease that cleaves betweenthe second target protein and the tag.

In some embodiments, the fusion protein is capable of forming theprotein aggregates in aqueous buffer with salt concentrations betweenabout 0 and 500 mM KCl or NaCl, or with salt concentrations less thanany one of or between any two of about 50, 100, 150, 200, 250, 300, 350,400, 450, and 500 mM KCl or NaCl. In some embodiments, the fusionprotein is capable of forming the protein aggregates when the fusionprotein is present in an aqueous solution at a concentration of lessthan about 5 μM or at a concentration of less than any one of or betweenany two of about 5, 10, 15, 20, 25, 30, 35, 40, 45, and 50 μM. In someembodiments, the fusion protein is also capable of forming the proteinaggregates when the fusion protein is present in an aqueous solution ata concentration of greater than 50 μM.

In some embodiments, the target protein (or first target protein) in afusion protein described above is ferritin or a ferritin subunit, afluorescent protein, an antibody, an antibody fragment, protein A,streptavidin, protein G, protein A/G, protein L, StrepTactin, anti-HAantibody (IgG1 against YPYDVPDYA (SEQ ID NO:15)), anti-cMYC antibody(IgG1 against EQKLISEEDL (SEQ ID NO:16)), anti-Glutathionc S-transfcrascantibody (GSTs), anti-FLAG antibody (e.g., anti-DYKDDDDK (SEQ ID NO:17)or anti-DDDDK (SEQ ID NO:18) antibody), a monobody, or an affinityclamp. In some embodiments, the target protein is an antibody fragment(e.g., Fab or scFv), monobody, or affinity clamp that specifically bindsto cMYC, GST, FLAG, or other protein “tags” known to those of skill inthe art.

In some embodiments, the target protein is ferritin. Ferritin is aprotein expressed in many living organisms that stores iron and releasesit in a controlled fashion. In some embodiments, the ferritin causes theprotein aggregates to be paramagnetic, which allows them to be pelleted,manipulated, or removed using magnets. The ferritin in the proteinaggregates described herein can be from any species. It is within thecapability of a person of ordinary skill in the art to select a ferritinprotein to include in the protein aggregates and to select a suitablenucleic acid encoding a ferritin protein or subunit. In someembodiments, the fusion protein comprised in the protein aggregatefurther comprises the ferritin. That is, the fusion protein can includethe self-assembly domain, a target protein, and ferritin or a ferritinsubunit. The fusion protein can also comprise only the self-assemblydomain and ferritin. In some embodiments, the ferritin is comprised in asecond fusion protein comprising a self-assembly domain. In someembodiments, the protein aggregate further comprises a fluorescentprotein. In some embodiments, the fusion protein further comprises thefluorescent protein. In some embodiments, the fluorescent protein iscomprised in a second fusion protein comprising a self-assembly domain.In some embodiments, the fluorescent protein is Clover or mRuby2. Insome embodiments, the protein aggregate further comprises self-assemblydomain proteins fused to other protein components. The self-assemblydomain proteins can be naturally occurring proteins that contain aself-assembly domain or isolated self-assembly domain sequences. It iscontemplated that in some embodiments, the protein aggregate does notcontain any naturally-occurring proteins. In some embodiments, themajority of the fusion protein molecules that comprise the aggregateform direct noncovalent protein-protein interactions with other fusionprotein molecules. In some embodiments, the protein-protein interactionsare aromatic-aromatic, cation-aromatic, or hydrophobic interactions.

In some embodiments, a fusion protein as described above is comprised inan aqueous composition comprising an aggregate nucleating agent. In someembodiments, the aggregate nucleating agent is a thermally unstableprotein that unfolds or misfolds at temperatures at or below about 40,42, 45, or 50° C. In some embodiments, the aggregate nucleating agent isfirefly luciferase.

Also disclosed is a self-assembly domain covalently conjugated to one ormore other polypeptides through a non-peptide bond. The self-assemblydomain and the other polypeptide can be separately expressed and thenconjugated together through chemical cross-linking means, which areknown to persons of skill in the art. The resulting molecule can be usedto form protein aggregates according to the methods described herein.

In some embodiments, any of the fusion proteins described above cancomprise a second, third, fourth, or fifth target protein or more.

In some embodiments, it is contemplated that a fusion protein consistsentirely of a contiguous string of amino acids. In some embodiments, thefusion protein does not have any additional chemical entity joined tothe amino acid string. It is also contemplated that the fusion proteincan consist of only a self-assembly domain and a target protein as asingle, contiguous amino acid string.

In some embodiments, a fusion protein described above can be conjugatedto another polypeptide or other molecule through peptide or non-peptidecovalent bonds. The self-assembly domain itself can also be conjugatedto other polypeptides or other types of molecules through peptide ornon-peptide covalent bonds. For example, in some embodiments, theself-assembly domain can be conjugated to biotin. In some embodiments,the self-assembly domain is not part of a fusion protein with a targetprotein, but is covalently conjugated to another molecule. The othermolecule can include other polypeptides, small molecules, nucleic acids,or other types of molecules.

In some embodiments, the invention comprises a method of delivering asubstance to a specific body site, comprising, (a) providing a fusionprotein comprising a target protein capable of binding the substancefused to a heat-inducible self-assembly domain, (b) adding the substanceto the fusion protein, (c) administering the fusion protein andsubstance to a patient, and (d) locally heating the body site. Inspecific embodiments, the substance is a nucleic acid, a protein orpeptide-based therapeutic.

In some embodiments, the invention comprises a method of assessingmodulators of aggregation in vitro by (a) providing a heat-inducibleself-assembly domain in an aqueous solution, (b) adding a substance, (c)administering heat to the sample, and (d) measuring the degree ofaggregation of the domain.

In some embodiments, the invention comprises a method of assessingmodulators of aggregation in vitro by (a) providing a heat-inducibleself-assembly domain fused to a protein in an aqueous solution, (b)adding a substance, (c) administering heat to the sample (d) measuringthe degree of aggregation of the fusion protein. In specificembodiments, the protein is a fluorescent protein. In other specificembodiments, a mixture of fusion proteins is used where each protein hasa heat-inducible self-assembly domain and a fluorescent protein that caninteract with other fusion proteins in the mixture to provide FRETmediated fluorescence upon assembly.

In some embodiments, the invention comprises a method of assessingmodulators of aggregation in vivo by (a) expressing a heat-inducibleself-assembly domain fused to a protein in a cell, (b) administering asubstance to the cell, (c) administering heat to the sample, (d)measuring the degree of aggregation of the fusion protein. In specificembodiments, the domain is a fluorescent protein. In other specificembodiments a mixture of fusion proteins is expressed where each proteinhas a heat-inducible self-assembly domain and a fluorescent protein thatcan interact with other fusion proteins in the mixture to provide FRETmediated fluorescence upon assembly.

It is contemplated that any embodiment described herein can be combinedwith any other described embodiment. For example, the features describedfor protein aggregates or fusion proteins in one embodiment can beapplied to the protein aggregate or fusion proteins of any otherembodiment. Likewise, any method steps described in a given method canbe included in any other described method, and any method canincorporate or use any fusion protein or protein aggregate describedherein.

As used herein, a “self-assembly domain” is a polypeptide sequence thatimparts to a polypeptide, of which it is a part of, the ability to formprotein aggregates under certain conditions. In some embodiments, theself-assembly domain is heat-inducible; that is, a protein that includesa heat-inducible self-assembly domain is soluble in aqueous solution atrelatively low temperatures (e.g., below about 35° C.) but assemblesinto aggregates with other proteins that include a heat-inducibleself-assembly domain upon heating to a higher temperature (e.g., atleast about 35° C.).

As used herein, “target protein” means a polypeptide that is distinctfrom a self-assembly domain. The term “target protein” excludes theamino acids that make up the self-assembly domain itself. The term“target protein” also excludes polypeptides that naturally possess aself-assembly domain, such as some GST-like proteins from Saccharomycescerevisiae or Ogataea parapolymorpha.

As used herein, a “fusion protein” is a single, contiguous polypeptidemolecule that comprises two or more distinct amino acid sequencesderived from at least two distinct sources. In some embodiments, adistinct source can be a naturally-occurring gene product sequence, aman-made polypeptide sequence, or fragments of either. In someembodiments, each distinct amino acid sequence included in a fusionprotein has at least or at most 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100amino acids or any derivable range therein. In some embodiments, eachdistinct amino acid sequence has a distinct function that is associatedwith its source and includes enough of the amino acid sequence from thesource to impart that functionality to the fusion protein. For example,in some embodiments, a fusion protein includes an amino acid sequencederived from a self-assembly domain and an amino acid sequence derivedfrom a green fluorescent protein. In such a fusion protein, theself-assembly domain amino acid sequence has self-assembly functionalityand the green fluorescent protein amino acid sequence has fluorescencefunctionality. The functionality imparted to the fusion protein by thedistinct amino acid sequence may also be, for example, binding to aspecific protein, small molecule, or ligand; performing a structuralrole; undergoing a conformation change under certain conditions;performing an enzymatic function such as catalyzing a chemical reaction;fluorescing under certain conditions; and so forth. In some embodimentsdescribed herein, the fusion proteins comprise distinct amino acidsequences from more than two distinct sources. A fusion protein caninclude at least, at most, or exactly 2, 3, 4, 5, 6, 7, or more distinctproteins or polypeptides (or any derivable range therein). Theself-assembly domain in the fusion proteins described herein may bederived from a naturally-occurring protein sequence or may beartificial. As used herein, “fusion protein” does not include apolypeptide that is wholly derived from a single, naturally occurringgene product. As used herein, “fusion proteins” are notnaturally-occurring.

The terms “a” and “an” are defined as one or more unless this disclosureexplicitly requires otherwise.

The term “substantially” is defined as being largely but not necessarilywholly what is specified (and include wholly what is specified) asunderstood by one of ordinary skill in the art. In any disclosedembodiment, the term “substantially” may be substituted with “within [apercentage] of” what is specified, where the percentage includes 0.1, 1,5, and 10 percent.

The terms “comprise” (and any form of comprise, such as “comprises” and“comprising”), “have” (and any form of have, such as “has” and“having”), “include” (and any form of include, such as “includes” and“including”) and “contain” (and any form of contain, such as “contains”and “containing”) are open-ended linking verbs. As a result, the methodsand systems of the present invention that “comprises,” “has,” “includes”or “contains” one or more elements possesses those one or more elements,but is not limited to possessing only those one or more elements.Likewise, an element of a method or system of the present invention that“comprises,” “has,” “includes” or “contains” one or more featurespossesses those one or more features, but is not limited to possessingonly those one or more features.

Throughout this application, the term “about” is used to indicate that avalue includes the standard deviation of error for the device or methodbeing employed to determine the value.

Furthermore, a structure that is capable performing a function or thatis configured in a certain way is capable or configured in at least thatway, but may also be capable or configured in ways that are not listed.Metric units may be derived from the English units provided by applyinga conversion and rounding to the nearest millimeter.

The feature or features of one embodiment may be applied to otherembodiments, even though not described or illustrated, unless expresslyprohibited by this disclosure or the nature of the embodiments.

Any method or system of the present invention can consist of or consistessentially of rather than comprise/include/contain/have any of thedescribed elements and/or features and/or steps. Thus, in any of theclaims, the term “consisting of” or “consisting essentially of” can besubstituted for any of the open-ended linking verbs recited above, inorder to change the scope of a given claim from what it would otherwisebe using the open-ended linking verb.

Details associated with the embodiments described above and others arepresented below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Overview of purification method. Recombinant target protein(oval) linked to a temperature-sensitive glutathione-S-transferase(GST)-like polypeptide tag (tsG tag) (square) by a protease-cleavablelinker (triangle) is expressed in host cells, which are lysed to releasethe protein and many contaminants. Soluble lysate is incubated for 10minutes at 50° C. and centrifuged at 20,000 g. The pellet is resuspendedand incubated with a tsG-tagged protease, which cleaves the linker andliberates the target protein. After another 10 minutes 50° C. incubationand centrifugation, the purified, tag-free protein is recovered from thesupernatant.

FIG. 2. Purification of target protein mRuby2 (red fluorescent protein)from crude E. coli lysate, illustrated on an SDS-PAGE gel developed withCoomassie stain. Here, the protease is tsG-TEV, tobacco etch virusprotease, which cleaves the seven-amino-acid sequence ENLYFQS (SEQ IDNO:19) between Q and S, leaving a single serine residue attached to thetarget protein. The target protein is mRuby2, a red fluorescent proteinvariant. Lane 1, E. coli lysate supernatant after centrifuging out celldebris. Lanes 2-5, total (T) protein, supernatant (S), wash (W), andpellet (P) material after first 50° C./10 min treatment andcentrifugation at 17,000 g for 10 minutes. Lanes 6-8: supernatant, wash,and pellet material after 2 h incubation with added tsG-TEV protease andsecond 50° C./10 min treatment. Lane 6 contains substantially puremRuby2 of the expected molecular weight.

FIG. 3. Detailed description of the purification scheme described inFIG. 1. A, Protein components. The target protein is expressed as afusion protein with a cleavable temperature-sensitive GST-like tag (tsGtag). A protease capable of cleaving the tag is purified once bystandard affinity methods. B, Details of the method. Soluble lysatecontaining the tagged target protein is passed through a set of steps togenerate soluble, tag-free target protein. No affinity chromatography isemployed. The method can be completed in under three hours and requiresonly a benchtop centrifuge.

FIG. 4A-4B. FIG. 4A shows the purification of the fluorescent protein,Clover. FIG. 4B compares purification of six different proteins (Clover,mRuby2, hGH, Suil, β-Gal, Pab1) using the current method withpurification using the traditional His-tag method. The top bar graph ofFIG. 4B shows the purity achieved for each protein using the FENEXpurification method described herein (left bar in each pair) and theHis-tag purification method (right bar in each pair). The bottom bargraph of FIG. 4B shows the yield (in mg of protein per liter of culture)achieved for each protein using the FENEX purification method (left barin each pair) and the His-tag purification method (right bar in eachpair). The purification method described herein is twice as fast asHis-tag purification, allows for simultaneous purification of manyproteins (high-throughput screens) and is inexpensive.

FIG. 6. Recombinant yeast proteins rapidly and autonomouslyself-assemble into large particles in vitro, recapitulating in vivo TRAPformation. A, Sizing gel of purified proteins. B, Thermally-triggeredself-assembly of purified proteins monitored by absorbance; temperaturechange at t=0. C, Comparison of in vivo and in vitro results after twominutes at the indicated temperature.

FIG. 7. GST-like domains likely mediate thermal assembly in the AMEcomplex and in the eEF-1B complex. A, Domain architecture. Rectanglesshow GST-like domains, circles show GST-N(thioredoxin-like) subdomains,and squares show GST-C subdomains. B, Ternary complex of AME GST-likeinteraction domains. C, Rapid response of proteins in A to thermal shiftmonitored by mass-spectrometric analysis.

FIG. 8 shows that purified AME complex (complex of three proteins:aminoacylation cofactor (Arc1), methionyl-tRNA synthetase (Mes1), andglutamyl-tRNA synthetase (Gus1)) forms large aggregates upon heat shock.Shown on the left panel is the elution profile from Superdex200 gelfiltration column. Above the elution profile is a western blotdemonstrating the trimeric complex eluted in fraction 12. Thisdemonstrates reconsititution of a stoichiometric three-protein complex.Shown on the right panel is a dynamic light scattering plot whichdemonstrates the size distribution profile of the individual proteins insolution and of the AME complex at the indicated temperatures. Gus1, andMes1 rapidly form large assemblies when heated (dynamic light scatteringdata), whereas Arc1 shows only modest assembly. Suil, another yeastprotein, shows no assembly, as a negative control.

FIG. 9 shows that virtually all of the AME complex is assembled afterincubation for 15 minutes at 46° C. Shown is the elution profile fromSuperose 6 size exclusion chromatography of soluble (non-pelletable)material. At 15 minutes, 46° C., the majority of the protein elutes in 1mL volume, indicating assembly of large AME complexes. The tallest peak(the left-most peak) is soluble assembly. The second tallest peak showsAME incubated at room temperature (25° C.) for comparison.

FIG. 10 shows that AME remains functional and possesses normal fidelityafter heat shock at 46° C. for 15 min. Aminoacylation of tRNA^(Met) with³⁵S methionine is used as a functional readout of AME activity. Topleft, kinetic assay comparing equimolar amounts of unheated AME (top,straight line), heated AME (middle line), and unheated Mes1 alone (lowerline) reveals that heated AME has slower kinetics relative to unheatedAME, yet retains higher activity than unheated Mes1 alone. Bars showresults of endpoint activity assay. Right, heated AME forms largecomplexes which pellet after centrifugation, and silver staining revealsthat the three AME components retain 1:1:1 stoichiometry in the pellet,suggesting the complex remains intact. Bottom, tRNA microarray-basedmisacylation assay (cf. Netzger et al. (2009), “Innate immune andchemically triggered oxidative stress modifies translational fidelity,”Nature 462:522-526). The differences between Mes1, AME, and heat-shockedAME methionine acylation patterns are minimal, indicating minimalperturbation of fidelity and again demonstrating activity ofheat-shocked AME.

FIG. 11 shows that Gus1N (N-terminus of Gus1; also called the tsGdomain) retains significant structure at assembly temperatures (50° C.).Data show circular dichroism spectra at 30° C. (lines with diamondpoints) and 50° C. (lines with circle points). Top, full-length Gus1remains folded and shows a temperature-dependent conformational change.Middle: Gus1ΔN, lacking N-terminal GST-like domain, shows almost nochange in response to temperature. Bottom, Gus1N, the isolated GST-likedomain, is very well-structured (gray line shows full-length Gus1 forcomparison), and displays a substantial temperature-dependentconformational change. Compare near-complete loss of structure with 6MGdn (guanidinium HCl) and 95° C. heating, which denature Gus1N.

FIG. 12 shows that assembly of Gus1 is domain-specific. Shown on theleft is a static light scattering plot showing that Gus1ΔN mutant, whichlacks the N terminal GST-like domain, does not assemble into largecomplexes after a temperature shift from 30 to 50° C. In contrast, Gus1N(a polypeptide consisting only of the the N-terminal GST-like domain)assembles into large aggregates rapidly (about 30-60 seconds) after a 30to 50° C. temperature shift.

FIG. 13 shows that Yef3 rapidly forms large particles in response to atemperature shift from room temperature to 50° C. (solid line), 46° C.(dashed), and 42° C. (dot-dash), but remains unassembled at 30° C.(dotted).

FIG. 14A-B shows the Gus1N self-assembling domain. Shown in A is ascheme which depicts the self-assembly of the Gus1N domains. Shown in Bis the Gus1N-affinity domain fusion protein.

FIG. 15 depicts a purification scheme using the methods of thedisclosure. This purification scheme is further described in Example 3.Briefly, the scheme depicts the steps: (1) heat-shock Gus1N-PDZ for 10minutes at 48° C.; (2) pellet and mix with myosin V(MV)-containinglysate; (3) incubate together to let MV bind to PDZ; (4) centrifuge anddiscard supernatant (contaminants); (5) add the elution peptide whichoutcompetes c-tagged MV and releases it from PDZ; (6) centrifuge,product is purified MV in the supernatant and Gus1N-PDZ in the pellet.

FIG. 16A-B shows Gus1N-PDZ expression (A) and purification (B) using aNi column. Gus1N-PDZ protein expresses very well under standardconditions, namely OD=0.6, 1 mM IPTG at 30° C. and a good level ofexpression is achieved after about 4 hours Only one-step purificationwas required to purify the protein. A Ni column was used for thepurification, and the average yield from two separate purifications is17.53 mg/L of cell culture.

FIG. 17A-B shows the specificity of the Gus1N-PDZ for the target. Shownin A is a cartoon depiction of the specificity assay. Heat-shockedGus1N-PDZ was incubated with either Clover-C or tagless Clover, thepellets were washed, and then the pellets were visualized by using bothUV and GFP channels. Shown in B are the results that demonstrate thatGus1N-PDZ binds specifically to clover-Ctag and not untagged clover(second and third panel showing UV and GFP results). The only pellet toemit any light is the one incubated with tagged Clover.

FIG. 18 demonstrates that Gus1N-PDZ binds its targets specifically. Thisfigure is similar to FIG. 17, however, elution was performed using theelution peptide in this experiment. Each sample was split into threefractions which are resin after elution, supernatant (fraction not boundto Gus1NPDZ), and eluted fractions. As can be seen in the figure,nothing eluted in case of tagless Clover while in the case of C-taggedClover there is got eluted Clover-C in the eluted fraction. Comparingthe lanes for Gus1N-PDZ—elution, Gus1N-PDZ+Clover—elution, andGus1N-PDZ+Clover-Ctag—elution, only the last elution lane had the Cloverprotein. Therefore, the Gus1N-PDZ provides for a resin with little or nobackground contamination. These results are consistent with previousresults (e.g., FIG. 17). The Gus1N-PDZ used was 583 μg, the Clover orClover-Ctag used was 100 μL of 30 μM. The peptide used was 100 μL of 200μM.

FIG. 19 shows the purification of Clover-Ctag using Gus1N-PDZ accordingto the methods described in Example 3. This experiment was done todetermine if Gus1N-PDZ could be used to purify out a target protein froma complex lysate. Clover-C was expressed in bacteria, and the lysate waspre-shocked with Gus1N-PDZ. Following this, the elution was performed.The Gus1N-PDZ used was 583 μg, the Clover or Clover-Ctag lysate used was100 μL. The peptide used was 100 μL of 200 μM. Clover-C was successfullypurified from the lysate with just some residual Gus1N-PDZ.

FIG. 20 depicts the purification of Myosin V using the methods describedin Example 3 compared to traditional methods using anti-FLAG resin. TheGus1N-PDZ used was 13 mg for the whole preparation. 250 mL of crudeextract was used. The peptide used was 600 μL of 400 μM. GEAR(Genetically-Encoded Affinity Resin) refers to the Gus1N-PDZ resin. Ascan be seen in the elution lanes, MV was purified in an amount that wascomparable to purification using an anti-FLAG tag method. The amount ofMV purified by the Gus1N-PDZ method was 25% less, however, the degree ofpurity was higher, and the capacity of the resin was higher, since alarge portion of uneluted MV can be seen.

FIG. 21—The elution peptide (EP) for competing Ctag-tagged protein offof Gus1N-PDZ resin may be produced using the Gus1N system. AGus1N-tevC-EP construct is expressed in E. coli, and purified usingheat/centrifugation as described in Example 2. The elution peptide maythen be used as in Example 3.

DETAILED DESCRIPTION OF THE INVENTION

Various features and advantageous details are explained more fully withreference to the non-limiting embodiments that are illustrated in theaccompanying drawings and detailed in the following description. Itshould be understood, however, that the detailed description and thespecific examples, while indicating embodiments of the invention, aregiven by way of illustration only, and not by way of limitation. Varioussubstitutions, modifications, additions, and/or rearrangements willbecome apparent to those of ordinary skill in the art from thisdisclosure.

In the following description, numerous specific details are provided toprovide a thorough understanding of the disclosed embodiments. One ofordinary skill in the relevant art will recognize, however, that theinvention may be practiced without one or more of the specific details,or with other methods, components, materials, and so forth.

A. PROTEINS AND PROTEIN EXPRESSION 1. Protein Sequences

In some embodiments, fusion proteins and/or protein aggregates describedherein include self-assembly domain sequences from a GST-like domain, asdescribed herein or from the proteins set forth in the table below:

Common Systematic Name Name YGL105W Arc1 YGR264C Mes1 YGL245W Gus1YPL048W Tef3 YKL081W Tef4 YAL003W Efb1, YLR249W Yef3 YBL039C Ura7

The table above gives the systematic name from S. cerevisiae, but it iscontemplated that the homolog from other species may be used.

In some embodiments, the fusion proteins and/or protein aggregatesdescribed herein include fluorescent proteins. One fluorescent proteinthat can be used is Clover, a sequence for which is set forth in GenBankaccession number AFR60231, which is hereby incorporated by reference.Further fluorescent proteins are described in Lee et al. (PLoS One8:367902 (2013)), which is hereby incorporated by reference.

2. Polypeptide Production

In specific embodiments, all or part of proteins described herein canalso be synthesized in solution or on a solid support in accordance withconventional techniques. Various automatic synthesizers are commerciallyavailable and can be used in accordance with known protocols. See, forexample, Stewart and Young, (1984); Tam et al., (1983); Merrifield,(1986); and Barany and Merrifield (1979). Alternatively, recombinant DNAtechnology may be employed wherein a nucleotide sequence that encodes apeptide or polypeptide is inserted into an expression vector,transformed or transfected into an appropriate host cell and cultivatedunder conditions suitable for expression.

One embodiment includes the use of gene transfer to cells, includingmicroorganisms, for the production and/or presentation of proteins. Thegene for the protein of interest may be transferred into appropriatehost cells followed by culture of cells under the appropriateconditions. A nucleic acid encoding virtually any polypeptide may beemployed. The generation of recombinant expression vectors, and theelements included therein, can be performed by routine techniques knownto those of skill in the art.

In some embodiments, fusion proteins can be expressed from a nucleotideconstruct that encodes the entire fusion protein. Alternatively, fusionproteins can be formed by covalently joining different proteins afterthey have already been produced.

3. Protein Purification or Isolation

In certain embodiments a protein or peptide or a composition comprisingsuch a protein or peptide may be isolated or purified. Proteinpurification techniques are well known to those of skill in the art.These techniques involve, at one level, the homogenization and crudefractionation of the cells, tissue or organ in to polypeptide andnon-polypeptide fractions. The protein or polypeptide of interest may befurther purified using chromatographic and electrophoretic techniques toachieve partial or complete purification (or purification tohomogeneity). Analytical methods particularly suited to the preparationof a pure peptide are ion-exchange chromatography, gel exclusionchromatography, polyacrylamide gel electrophoresis, affinitychromatography, immunoaffinity chromatography and isoelectric focusing.An example of receptor protein purification by affinity chromatographyis disclosed in U.S. Pat. No. 5,206,347, the entire text of which isincorporated herein by reference. A particularly efficient method ofpurifying peptides is fast performance liquid chromatography (FPLC) oreven high performance liquid chromatography (HPLC).

A purified protein or peptide is intended to refer to a composition,isolatable from other components, wherein the protein or peptide ispurified to any degree relative to its naturally-obtainable state. Anisolated or purified protein or peptide, therefore, also refers to aprotein or peptide free from the environment in which it may naturallyoccur.

Generally, “purified” will refer to a protein or peptide compositionthat has been subjected to fractionation to remove various othercomponents, and which the composition substantially retains itsexpressed biological activity. Where the term “substantially purified”is used, this designation will refer to a composition in which theprotein or peptide forms the major component of the composition, such asconstituting about 50%, about 60%, about 70%, about 80%, about 90%,about 95%, or more of the proteins in the composition.

A peptide, polypeptide or protein that is “purified to homogeneity,” asapplied to the present invention, means that the peptide, polypeptide orprotein has a level of purity where the peptide, polypeptide or proteinis substantially free from other proteins and biological components. Forexample, a purified peptide, polypeptide or protein will often besufficiently free of other protein components so that degradativesequencing may be performed successfully.

Various methods for quantifying the degree of purification of theprotein or peptide are known to those of skill in the art in light ofthe present disclosure. These include, for example, determining thespecific activity of an active fraction, or assessing the amount ofpolypeptides within a fraction by SDS/PAGE analysis. A particular methodfor assessing the purity of a fraction is to calculate the specificactivity of the fraction, to compare it to the specific activity of theinitial extract, and to thus calculate the degree of purity therein,assessed by a “-fold purification number.” The actual units used torepresent the amount of activity will, of course, be dependent upon theparticular assay technique chosen to follow the purification, andwhether or not the expressed protein or peptide exhibits a detectableactivity.

To purify a desired protein, polypeptide, or peptide a natural orrecombinant composition comprising at least some specific proteins,polypeptides, or peptides may be subjected to fractionation to removevarious other components from the composition. Various techniquessuitable for use in protein purification are well known to those ofskill in the art. These include, for example, precipitation withammonium sulfate, PEG, antibodies and the like, or by heat denaturation,followed by: centrifugation; chromatography steps such as ion exchange,gel filtration, reverse phase, hydroxylapatite and affinitychromatography; isoelectric focusing; gel electrophoresis; andcombinations of these and other techniques. As is generally known in theart, it is believed that the order of conducting the variouspurification steps may be changed, or that certain steps may be omitted,and still result in a suitable method for the preparation of asubstantially purified protein or peptide.

Another example is the purification of a specific fusion protein using aspecific binding partner. Such purification methods are routine in theart. Certain aspects of the present invention provide DNA sequences forthe specific proteins, and any fusion protein purification method may bepracticed. However, given many DNA and proteins are known, or may beidentified and amplified using the methods described herein, anypurification method can now be employed.

There is no general requirement that the protein or peptide always beprovided in their most purified state. Indeed, it is contemplated thatless substantially purified products will have utility in certainembodiments. Partial purification may be accomplished by using fewerpurification steps in combination, or by utilizing different forms ofthe same general purification scheme. For example, it is appreciatedthat cation-exchange column chromatography performed utilizing an HPLCapparatus will generally result in a greater “-fold” purification thanthe same technique utilizing a low pressure chromatography system.Methods exhibiting a lower degree of relative purification may haveadvantages in total recovery of protein product, or in maintaining theactivity of an expressed protein.

Affinity chromatography is a chromatographic procedure that relies onthe specific affinity between a substance to be isolated and a moleculeto which it can specifically bind. This is a receptor-ligand type ofinteraction. The column material is synthesized by covalently couplingone of the binding partners to an insoluble matrix. The column materialis then able to specifically adsorb the substance from the solution.Elution occurs by changing the conditions to those in which binding willnot occur (e.g., altered pH, ionic strength, temperature, etc.). Thematrix should be a substance that itself does not adsorb molecules toany significant extent and that has a broad range of chemical, physicaland thermal stability. The ligand should be coupled in such a way as tonot affect its binding properties. The ligand should also providerelatively tight binding. And it should be possible to elute thesubstance without destroying the sample or the ligand.

B. SEQUENCE LISTING

Systematic Name Common Name YGL105W Arc1 YGR264C Mes1 YGL245W Gus1YPL048W Tef3 YKL081W Tef4 YAL003W Efb1, YLR249W Yef3 YBL039C Ura7

S. cerevisiae YGL105W/Arc1:

(SEQ ID NO: 1) MSDLVTKFESLIISKYPVSFTKEQSAQAAQWESVLKSGQIQPHLDQLNLVLRDNTFIVSTLYPTSTDVHVFEVALPLIKDLVASSKDVKSTYTTYRHILRWIDYMQNLLEVSSTDKLEINHDLDLPHEVIEKKKKAPAGGAADAAAKADEDVSKKAKKQDHPRGKPDEETLKKLREEAKAKKAAKKAANAKQQQEQQNKAPEKPKPSAIDFRVGFIQKAIKHPDADSLYVSTIDVGDEEGPRTVCSGLVKHFPLDAMQERYVVVVCNLKPVNMRGIKSTAMVLCGSNDDKVEFVEPPKDSKAGDKVFFEGFGDEAPMKQLNPKKKIWEHLQPHFTTNDGLEVI FKDEEEKDHPVRKLTNAKGESFKVASI ANAQVR*

S. cerevisiae YGR264C/Mes1:

(SEQ ID NO: 2) MSFLISFDKSKKHPAHLQLANNLKIALALEYASKNLKPEVDNDNAAMELRNTKEPFLLFDANAILRYVMDDFEGQTSDKYQFALASLQNLLYHKELPQQHVEVLTNKAIENYLVELKEPLTTTDLILFANVYALNSSLVHSKFPELPSKVHNAVALAKKHVPRDSSSFKNIGAVKIQADLTVKPKDSEILPKPNERNILITSALPYVNNVPHLGNIIGSVLSADIFARYCKGRNYNALFICGTDEYGTATETKALEEGVTPRQLCDKYHKIHSDVYKWFQIGFDYFGRTTTDKQTEIAQHIFTKLNSNGYLEEQSMKQLYCPVHNSYLADRYVEGECPKCHYDDARGDQCDKCGALLDPFELINPRCKLDDASPEPKYSDHIFLSLDKLESQISEWVEKASEEGNWSKNSKTITQSWLKDGLKPRCITRDLVWGTPVPLEKYKDKVLYVWFDATIGYVSITSNYTKEWKQWWNNPEHVSLYQFMGKDNVPFHTVVFPGSQLGTEENWTMLHHLNTTEYLQYENGKFSKSRGVGVFGNNAQDSGISPSVWRYYLASVRPESSDSHFSWDDFVARNNSELLANLGNFVNRLIKFVNAKYNGVVPKFDPKKVSNYDGLVKDINEILSNYVKEMELGHERRGLEIAMSLSARGNQFLQENKLDNTLFSQSPEKSDAVVAVGLNIIYAVSSIITPYMPEIGEKINKMLNAPALKIDDRFHLAILEGHNINKAEYLFQRIDEKKIDEWRAKYGGQQ V*

S. cerevisiae YGL245W/Gus1:

(SEQ ID NO: 3) MPSTLTINGKAPIVAYAELIAARIVNALAPNSIAIKLVDDKKAPAAKLDDATEDVFNKITSKFAATFDNGDKEQVAKWVNLAQKELVIKNFAKLSQSLETLDSQLNLRTFTLGGLKYSAADVACWGALRSNGMCGSIIKNKVDVNVSRWYTLLEMDPIFGEAHDFLSKSLLELKKSANVGKKKETHKANFEIDLPDAKMGEVVTRFPPEPSGYLHIGHAKAALLNQYFAQAYKGKLIIRFDDTNPSKEKEEFQDSILEDLDLLGIKGDRITYSSDYFQEMYDYCVQMIKDGKAYCDDTPTEKMREERMDGVASARRDRSVEENLRIFTEEMKNGTEEGLKNCVRAKIDYKALNKTLRDPVIYRCNLTPHHRTGSTWKIYPTYDFCVPIVDAIEGVTHALRTIEYRDRNAQYDWMLQALRLRKVHIWDFARINFVRTLLSKRKLQWMVDKDLVGNWDDPRFPTVRGVRRRGMTVEGLRNFVLSQGPSRNVINLEWNLIWAFNKKVIDPIAPRHTAIVNPVKIHLEGSEAPQEPKIEMKPKHKKNPAVGEKKVIYYKDIVVDKDDADVINVDEEVTLMDWGNVIITKKNDDGSMVAKLNLEGDFKKTKHKLTWLADTKDVVPVDLVDFDHLITKDRLEEDESFEDFLTPQTEFHTDAIADLNVKDMKIGDIIQFERKGYYRLDALPKDGKPYVFFTIPDGKS VNKYGAKK*

S. cerevisiae YPL048W/Tef3:

(SEQ ID NO: 4) MSQGTLYANFRIRTWVPRGLVKALKLDVKVVTPDAAAEQFARDFPLKKVPAFVGPKGYKLTEAMAINYYLVKLSQDDKMKTQLLGADDDLNAQAQIIRWQSLANSDLCIQIANTIVPLKGGAPYNKKSVDSAMDAVDKIVDIFENRLKNYTYLATENISLADLVAASIFTRYFESLFGTEWRAQHPAIVRWFNTVRASPFLKDEYKDFKFADKPLSPPQKKKEKKAPAAAPAASKKKEEAKPAATETETSSKKPKHPLELLGKSTFVLDDWKRKYSNEDTRPVALPWFWEHYNPEEYSLWKVTYKYNDELTLTFMSNNLVGGFFNRLSASTKYMFGCLVVYGENNNNGIVGAVMVRGQDYVPAFDVAPDWESYDYAKLDPTNDDDKEFINNMWAWDKPVS VNGEPKEIVDGKVLK*

S. cerevisiae YKL081W/Tef4:

(SEQ ID NO: 5) MSQGTLYINRSPRNYASEALISYFKLDVKIVDLEQSSEFASLFPLKQAPAFLGPKGLKLTEALAIQFYLANQVADEKERARLLGSDVIEKSQILRWASLANSDVMSNIARPFLSFKGLIPYNKKDVDACFVKIDNLAAVFDARLRDYTFVATENISLGDLHAAGSWAFGLATILGPEWRAKHPHLMRWFNTVAASPIVKTPFAEVKLAEKALTYTPPKKQKAEKPKAEKSKAEKKKDEAKPADDAAPAKKPKHPLEALGKSTFVLDDWKRKYSNDDTRPVALPWFWEHYNPEEYSIWKVGYKYNDELTLTFMSNNLVGGFFNRLSASTKYMFGCLVVYGENNNNGIVGAVMVRGQDFAPAFDVAPDWESYEYTKLDPTKEEDKEFVNNMWAWDKPVVVNG EDKEIVDGKVLK*

S. cerevisiae YAL003W/Efb1:

(SEQ ID NO: 6) MASTDFSKIETLKQLNASLADKSYIEGTAVSQADVTVFKAFQSAYPEFSRWFNHIASKADEFDSFPAASAAAAEEEEDDDVDLFGSDDEEADAEAEKLKAERIAAYNAKKAAKPAKPAAKSIVTLDVKPWDDETNLEEMVANVKAIEMEGLTWGAHQFIPIGFGIKKLQINCVVEDDKVSLDDLQQSIEEDEDHVQSTDI AAMQKL*

S. cerevisiae YLR249W/Yef3:

(SEQ ID NO: 7) MSDSQQSIKVLEELFQKLSVATADNRHEIASEVASFLNGNIIEHDVPEHFFGELAKGIKDKKTAANAMQAVAHIANQSNLSPSVEPYIVQLVPAICTNAGNKDKEIQSVASETLISIVNAVNPVAIKALLPHLTNAIVETNKWQEKIAILAAISAMVDAAKDQVALRMPELIPVLSETMWDTKKEVKAAATAAMTKATETVDNKDIERFIPSLIQCIADPTEVPETVHLLGATTFVAEVTPATLSIMVPLLSRGLNERETGIKRKSAVIIDNMCKLVEDPQVIAPFLGKLLPGLKSNFATIADPEAREVTLRALKTLRRVGNVGEDDAIPEVSHAGDVSTTLQVVNELLKDETVAPRFKIVVEYIAAIGADLIDERIIDQQAWFTHITPYMTIFLHEKKAKDILDEFRKRAVDNIPVGPNFDDEEDEGEDLCNCEFSLAYGAKILLNKTQLRLKRARRYGICGPNGCGKSTLMRAIANGQVDGFPTQEECRTVYVEHDIDGTHSDTSVLDFVFESGVGTKEAIKDKLIEFGFTDEMIAMPISALSGGWKMKLALARAVLRNADILLLDEPTNHLDTVNVAWLVNYLNTCGITSITISHDSVFLDNVCEYIINYEGLKLRKYKGNFTEFVKKCPAAKAYEELSNTDLEFKFPEPGYLEGVKTKQKAIVKVTNMEFQYPGTSKPQITDINFQCSLSSRIAVIGPNGAGKSTLINVLTGELLPTSGEVYTHENCRIAYIKQHAFAHIESHLDKTPSEYIQWRFQTGEDRETMDRANRQINENDAEAMNKIFKIEGTPRRIAGIHSRRKFKNTYEYECSFLLGENIGMKSERWVPMMSVDNAWIPRGELVESHSKMVAEVDMKEALASGQFRPLTRKEIEEHCSMLGLDPEIVSHSRIRGLSGGQKVKLVLAAGTWQRPHLIVLDEPTNYLDRDSLGALSKALKEFEGGVIIITHSAEFTKNLTEEVWAVKDGRMTPSGHNWVSGQGAGPRIEKKEDEEDKFDAMGNKIAGGKKKKKLSSAELRKKKKERMKKKKELGDAYVSSDEEF*

S. cerevisiae YBL039C/Ura7:

(SEQ ID NO: 8) MKYVVVSGGVISGIGKGVLASSTGMLMKTLGLKVTSIKIDPYMNIDAGTMSPLEHGECFVLDDGGETDLDLGNYERYLGVTLTKDHNITTGKIYSHVIAKERKGDYLGKTVQIVPHLTNAIQDWIERVAKIPVDDTGMEPDVCIIELGGTVGDIESAPFVEALRQFQFKVGKENFALIHVSLVPVIHGEQKTKPTQAAIKGLRSLGLVPDMIACRCSETLDKPTIDKIAMFCHVGPEQVVNVHDVNSTYHVPLLLLEQKMIDYLHARLKLDEISLTEEEKQRGLELLSKWKATTGNFDESMETVKIALVGKYTNLKDSYLSVIKALEHSSMKCRRKLDIKWVEATDLEPEAQESNKTKFHEAWNMVSTADGILIPGGFGVRGTEGMVLAARWARENHIPFLGVCLGLQIATIEFTRSVLGRKDSHSAEFYPDIDEKNHVVVFMPEIDKETMGGSMRLGLRPTFFQNETEWSQIKKLYGDVSEVHERHRHRYEINPKMVDELENNGLIFVGKDDTGKRCEILELKNHPYYIATQYHPEYTSKVLDPSKPFLGLVAASAGILQDVIEGKYDLEAGENKFNF*

S. cerevisiae YGL245W/Gus1; 191Aa N-Terminal Polypeptide with GST-LikeDomain:

(SEQ ID NO: 9) MPSTLTINGKAPIVAYAELIAARIVNALAPNSIAIKLVDDKKAPAAKLDDATEDVFNKITSKFAAIFDNGDKEQVAKWVNLAQKELVIKNFAKLSQSLETLDSQLNLRTFILGGLKYSAADVACWGALRSNGMCGSIIKNKVDVNVSRWYTLLEMDPIFGEAHDFLSKSLLELKKSANVGKKKETHKANFE

S. cerevisiae YGR264C/Mes1; 207Aa N-Terminal Polypeptide with GST-LikeDomain:

(SEQ ID NO: 10) MSFLISFDKSKKHPAHLQLANNLKIALALEYASKNLKPEVDNDNAAMELRNTKEPFLLFDANAILRYVMDDFEGQTSDKYQFALASLQNLLYHKELPQQHVEVLTNKAIENYLVELKEPLTTTDLILFANVYALNSSLVHSKFPELPSKVHNAVALAKKHVPRDSSSFKNIGAVKIQADLTVKPKDSEILPKPNERNILI TSALPYV

S. cerevisiae YKL081W/Tef4; 156Aa N-Terminal Polypeptide with GST-LikeDomain:

(SEQ ID NO: 11) MSQGTLYINRSPRNYASEALISYFKLDVKIVDLEQSSEFASLFPLKQAPAFLGPKGLKLTEALAIQFYLANQVADEKERARLLGSDVIEKSQILRWASLANSDVMSNIARPFLSFKGLIPYNKKDVDACFVKIDNLAAVFDARLRDYTFV ATENIS

S. cerevisiae YPL048W/Tef3; 159Aa N-Terminal Polypeptide with GST-LikeDomain:

(SEQ ID NO: 12) MSQGTLYANFRIRTWVPRGLVKALKLDVKVVTPDAAAEQFARDFPLKKVPAFVGPKGYKLTEAMAINYYLVKLSQDDKMKTQLLGADDDLNAQAQIIRWQSLANSDLCIQIANTIVPLKGGAPYNKKSVDSAMDAVDKIVDIFENRLKNY TYLATENIS

(SEQ ID NO: 13) RGSIDTWV.

C-Tag:

(SEQ ID NO: 14) EEWETWV.

Elution Peptide:

Exemplary tags:

(SEQ ID NO: 15) YPYDVPDYA; (SEQ ID NO: 16) EQKLISEEDL; (SEQ ID NO: 17)DYKDDDDK and (SEQ ID NO: 18) DDDDK.

TEV Cleavage Site:

(SEQ ID NO: 19) ENLYFQS

C. EXAMPLES

The present invention will be described in greater detail by way ofspecific examples. The following examples are offered for illustrativepurposes only, and are not intended to limit the invention in anymanner. Those of skill in the art will readily recognize a variety ofnoncritical parameters which can be changed or modified to yieldessentially the same results.

Following a sudden increase in temperature, cells attenuate proteinsynthesis and mount the heat-shock transcriptional program, andeukaryotic cells additionally sequester proteins and RNA in stressgranules. How cells sense temperature remains unclear. Here, using anovel mass spectrometric method to identify protein aggregation at theproteome scale in budding yeast, Applicants show that within two minutesof a rise in temperature, a limited set of soluble proteins assemble invivo into large particles which are molecularly distinct from stressgranules. Remarkably, Applicants find that assembly isprotein-autonomous: recombinant, purified proteins self-assemble invitro with comparable kinetics in response to an equivalent thermalshift. For glutamyl-tRNA synthetase, autonomous thermal self-assemblyoccurs between stably folded proteins and reflects temperature-dependentconformational changes in specific protein-protein interaction domains.Applicants propose that a distributed system of sensor domains transducetemperature into autonomous protein assembly to effect rapid adjustmentof diffusible protein levels without transcription, translation, orprotein modifications.

Example 1 Self-Assembly Domains Induced by Heat to Form ProteinAggregates

Thermally-induced protein misfolding and aggregation have long beenthought to trigger the heat-shock response, but the sensitivity ofindividual proteins to thermal aggregation has remained unclear. Toexamine changes in protein aggregation in response to heat stress,Applicants used a proteome-scale mass spectrometric (MS) assay tomonitor the ratio of proteins found in the supernatant (aqueous-soluble)and pellet (aqueous-insoluble, detergent-soluble) fractions. To maximizeeffect sizes, Applicants targeted brief treatments after which only afraction of cells survive, shifting exponentially growing cells from 30°C. to 50° C. for two, four, and eight minutes. Applicants immediatelyharvested supernatant and 100,000 g pellet fractions from each, combinedthe supernatant fraction with the pellet fraction from cells grown onstable-isotope-labeled arginine and lysine, then analyzed the mixedsamples by liquid chromatography coupled to tandem mass spectrometry(LC-MS/MS). The data reveal that after two minutes, while the vastmajority of proteins showed no significant aggregation, a small set ofhighly soluble proteins form pelletable aggregates (FIG. 7C), with 31 of597 protein (5%) increasing at least four-fold in the insolublefraction. Little change was observed at later times, with Pearsoncorrelations ≥0.85 between supernatant/pellet ratios at 2, 4, and 8minutes.

These observations suggested a functional connection between theaggregating proteins, consistent with a thermally triggered assemblyprocess. Heat-induced stress granules were originally identified by asimilar centrifugation procedure. Indeed, most components of stressgranules induced by robust heat shock, previously studied by fluorescentimaging, are found in these rapidly assembling particles, with theexception of one protein, Dhh1, primarily associated with mRNAprocessing bodies (P bodies), and one small-subunit ribosomal protein.Stress granules are thought to coalesce around stalled 48S preinitiationcomplexes, in which the 40S small ribosomal subunit is a core component.Small-subunit proteins are universal markers for stress granules and theyeast ribosomal protein S30, but not large-subunit L25, accumulates inheat-induced granules. However, all of the 131 detected ribosomal geneproducts, including 56 from the small subunit, remained stronglyenriched in the supernatant and were entirely separable from theaggregating proteins at 2 min. Additional initiation-complex componentseIF-1A, eIF-2 (a, f3, and y subunits), and elF-1 also remained soluble.These and further results reported below indicate that these rapidlyforming particles do not co-assemble with preinitiation complexes orsmall ribosomal subunits, distinguishing them from stress granules. Theprocessing-body (P-body) markers Dcp2p also remained in the supernatant,distinguishing these particles from P-bodies. Applicants thereforedesignated them thermosensitive rapidly assembling particles (TRAPs).

The rapid formation of TRAPs, and their independence from preinitiationcomplexes led Applicants to wonder whether the constituent proteinsthemselves possessed the intrinsic ability to transduce a thermal shiftinto self-assembly. To test this possibility, Applicants purifiedseveral TRAP-forming proteins in recombinant form from bacteria (Yef3,Gus1, and CTP synthase/Ura7), along with two control proteins(Suil/eIF-1 and Hyp2/eIF-5A). Applicants suspended them in aqueousbuffer at concentrations approximating their physiological levels, andmonitored formation of large particles by visible-light absorbance at550 nm. When Applicants subjected each protein to the same 30° C. to 50°C. thermal shift in vitro, all TRAP-forming proteins rapidlyself-assembled into large particles, and the control proteins did not.Particles grew exponentially until exhaustion of unassembled material(FIG. 6B).

Self-assembly in vitro approached saturation in two minutes, consistentwith in vivo behavior (FIG. 6C). Applicants then asked whether lowertemperatures triggered assembly. Yef3 forms granules at 42° C. in vivowhich seed subsequent formation of genuine stress granules.Correspondingly, Yef3 self-assembled rapidly at 46° C. and 42° C. invitro with temperature-dependent kinetics (FIG. 13).

FIG. 8 shows that purified AME complex (complex of three proteins:aminoacylation cofactor (Arc1), methionyl-tRNA synthetase (Mes1), andglutamyl-tRNA synthetase (Gus1)) forms large aggregates upon heat shock.FIG. 9 shows that virtually all of the AME complex is assembled afterincubation for 15 minutes at 46° C. FIG. 10 shows AME complexes underelectron microscopy. The AME assemblies increase in abundance after 15min, 46° C., but are absent at 15 minutes, 30° C. FIG. 10 shows that AMEremains functional and possesses normal fidelity after heat shock at 46°C. for 15 min.

Applicant's data and previous work indicate that thermal self-assemblyin vitro recapitulates assembly in vivo with similartemperature-dependent kinetics.

Gus1 possesses a eukaryote-specific N-terminal domain, which has beenpreviously crystallized in isolation and shown to adopt aglutathione-S-transferase-like (GST-like) fold (FIG. 12). Applicantspurified this domain (Gus1N) and the remaining core synthetase domain(Gus1ΔN). Gus1ΔN did not form large particles at any tested temperatureup to 50° C. (FIG. 12), and showed a largely unperturbed structure (FIG.12). In contrast, Gus1N readily assembled in vitro (FIG. 12). Gus1N istherefore necessary and sufficient for thermal assembly in vitro.

Gus1N behaves like a thermometer, transducing a change in temperatureinto self-assembly. To determine the sensitivity of this phenomenon,Applicants turned to dynamic light scattering (DLS), which unlikeabsorbance is capable of resolving particles at the nanometer scale. DLSrevealed that this GST-like domain purified Gus1N adopts a GST-like foldand mediates binding of Gus1 to the cofactor Arc1, accelerating theaminoacylation rate of Gus1. In isolation, Gus1N rapidly assembled intolarge particles upon temperature shift (FIG. 12). Gus1N is thusnecessary and sufficient for full-length Gus1's temperature-dependentself-assembly in vitro.

H1,N15-HSQC NMR of Gus1N at 20° C. and 43° C. indicate that theenvironment of the amide groups in Gus1N are similar at the twotemperatures suggesting that self-assembly is mediated through a smallconformational change and that most of the structure remains foldedduring assembly.

To determine the consequences of thermal shift on protein structure,Applicants collected far-ultraviolet circular dichroism (CD) spectra.The CD spectrum of full-length Gust at 30° C. revealed a well-foldedstructure; at 50° C., the protein remained well-folded while losing somehelical structure (FIG. 11). The core synthetase, Gus1ΔN, showed minimalstructural change in response to the temperature shift (FIG. 11). Inmarked contrast, Gus1N underwent a significant conformational changeinvolving loss of helical structure, yet did so while preserving ahighly ordered, largely α-helical structure (FIG. 11), whereasdenaturation with 6M guanidine chloride (Gdn) disrupted Gus1N structureentirely (FIG. 11). These results closely match the in vitro assemblydata, showing that at the residue and oligomer levels, theeukaryote-specific Gus1N domain is a temperature-responsive elementlinked to a temperature-insensitive enzyme.

Applicants speculate that the formation of large aggregates after anear-lethal heat shock results from damage to the heat-sensing system. Asensory system's necessary sensitivity to a stimulus predisposes thatsystem to specific damage when the stimulus grows overwhelming, much aseyes are damaged by extremely bright light and ears by extremely loudsounds but not vice versa.

Repeated demonstrations that heat-shock-like responses can be generatedby protein misfolding at normal growth temperatures have led to thehypothesis that heat shock is signaled and sensed by misfolded proteins.Notably, however, it has never been established that thetemperature-induced misfolding of a native protein triggers the heatshock response. Applicants' results suggest the existence of analternative channel of information provided by domain-specificthermal-shift-induced assembly of sensory proteins. These resultsstrongly suggest that thermosensor-domain-mediated self-assembly, likelyoccurring in parallel in other proteins, is the first mechanistic stepconnecting a temperature change with stress-granule formation.

These results suggest a model in which environmental changes triggerproportionate changes in protein assembly, building up assembledproteins and reducing the population of freely diffusing proteins.Assembly may be reversible by cellular factors, or may require synthesisof new unassembled proteins. Applicants speculate that cellularchaperones, several of which are known to disaggregate misfoldedproteins, also disaggregate assemblies. If so, this would suggest aremarkably simple regulatory mechanism.

Example 2: Rapid, Low-Cost Purification of Recombinant Tag-Free Proteinswithout Affinity Chromatography (Fenex)

A method is described for purifying a wide range of recombinant proteinsemploying only mild heating (<50° C.) and centrifugation (<20,000 g)achievable with standard benchtop equipment.

Purification of proteins is essential to many biological and industrialpursuits, such as characterization of protein structure and function andthe development of drugs. Recombinant protein expression andpurification is a common strategy, because affinity tags optimized forselective binding to columns in affinity chromatography systems can beappended to the target protein, enabling use of the same system topurify many proteins. Affinity chromatography equipment remainsexpensive and complex, making protein purification inaccessible to many.

Certain proteins are routinely purified without affinity chromatography.A kilogram of RNAse A was famously purified by the Armour Co. by boilingbovine pancreas and centrifuging the resulting stew; RNAse A is the onlyprotein that remains in the supernatant after this treatment.Thermophilic proteins are often purified recombinantly from mesophilichosts (such as E. coli) by heat-denaturing the host lysate attemperatures intolerable to the host but tolerated by the thermophile.These examples remain rare exceptions.

Purification methods including removal of affinity tags typicallyrequire two separate purification steps. Typical generic proteinpurification systems require purchase or production of columns, affinityresin, and liquid-handling systems to control flow, measure propertiesof the flowing liquid, and collect fractions. A single separation oflysate containing affinity-tagged protein on an affinity column mostoften yields recombinant protein with the affinity tag still attached.Digestion with an affinity-tagged protease and a second separation isrequired to yield tag-free protein. The present method achieves bothseparation and tag removal without any affinity columns, resin, orliquid-handling systems, making it far simpler, faster, easier, andcheaper than common approaches.

A method for purifying a target protein is diagrammed in FIG. 1, and isdescribed below. The target protein is initially tagged and expressed inan arbitrary host organism; the method produces tag-free protein.

The method exploits a temperature-sensitive GST-like polypeptide (“tsGtag”) fused to two proteins: a target protein of interest, and aprotease. The tsG polypeptide has the property, discovered by Applicantsand as yet unreported, of rapidly self-assembling in response toincreases in temperature.

One embodiment comprises sequestering the target protein away fromsoluble host contaminants by heat-induced tsG-tag self-assembly,releasing the target protein from heat-aggregatable host contaminants byproteolysis with a tsG-tagged protease, and finally removingheat-aggregatable host contaminants, the protease, and the cleaved tagby heat-induced tsG self-assembly.

The self-assembly of tsG results in large particles which pellet readilyupon centrifugation at 10,000-20,000 g, attainable on a typical benchtopmicrocentrifuge.

FIG. 2 demonstrates purification of a test protein, the red fluorescentprotein mRuby2, from E. coli using one embodiment of the method.Briefly, E. coli were cultured and lysed using methods known to thoseskilled in the art, such as mechanical or chemical lysis. The solublelysate containing the fusion protein is heated for 10 minutes at 50degrees Celsius and then subjected to centrifugation for 2 minutes at17,000 g. The SDS-PAGE gel represents protein profile from samplesduring the process of the method. Lane 1 shows the starting materialafter cell lysis. Lanes 2-5, total (T) protein, supernatant (S), wash(W), and pellet (P) material after first 50° C./10 min treatment andcentrifugation at 17,000 g for 10 minutes. The supernatant comprisingsoluble contaminants was discarded. The remaining pellet comprisesinsoluble contaminants and the heat-assembled Gus1N-mRuby2. Next, asecond fusion protein comprising Gus1N and tobacco etch virus (TEV)protease was added to the remaining pellet, which was suspended in TEVcleavage buffer. The mixture was heated at 25 degrees Celsius for 2hours in which the TEV protease liberated the mRuby2 from the Gus1N. Themixture was then heated for 10 minutes at 50 degrees Celsius toaggregate mRuby2-free Gus1N protein. Insoluble contaminants and Gus1Nwere removed from mRuby2 by centrifugation for 2 minutes at 17,000 g.The soluble mRuby2 was decanted from the pellet. Lanes 6-8 of theSDS-PAGE in FIG. 2 shows the supernatant, wash, and pellet materialafter 2 h incubation with added tsG-TEV protease and second 50° C./10min treatment. Lane 6 contains substantially pure mRuby2 of the expectedmolecular weight. FIG. 3 illustrates the steps involved at the molecularlevel. FIG. 4A shows the purification of the fluorescent protein,Clover, using the current embodiment. FIG. 4B compares purification ofsix different proteins (Clover, mRuby2, hGH, Suil, 13-Gal, Pab1) usingthe current embodiment with purification using the traditional His-tagmethod. Certain embodiments of the method described herein achievescomparable purity to the His-tag method.

Certain embodiments of this method allows protein purification morecheaply and rapidly, with less equipment and less effort, than anymethod of which Applicants are aware. They are applicable topurification of any soluble proteins, particularly for initial orhigh-throughput studies.

In addition to its simplicity, the method has proven to be unusuallygood at removing the protease and uncleaved fusion protein from thefinal purification. This is often a challenge for existing affinitypurification schemes, which, even with a second round of purification,fail to completely remove the uncleaved protein.

In principle, a very wide range of proteins are amenable to purificationby various embodiments of the invention.

Preferably, for certain embodiments, target proteins are stable attemperatures and durations necessary for assembly of the tsG domains.Reaction temperatures can be lowered by engineering of the tsG domain.In certain embodiments, however, such as the embodiment shown in FIG.19, the target protein is not heated to the temperature necessary forassembly of the tsG domains.

Preferably, for certain embodiments, target proteins are tolerant offusion to the tsG domain and protease cleavage site. Most affinitypurification methods require fusions of some sort (alternatives areantibodies and affinity reagents designed to be specific to the proteinof interest), and both the type of domain (GST-like) and the cleavagesite employed in Applicants' proof-of-concept experiment are widelyemployed in protein purification experiments.

Preferably, the target protein is soluble under the conditions employed.

For some applications, subsequent purification steps may be used.

Engineering a wide range of proteases by fusing them to the tsG tag willenable utilization of a wide range of protease cleavage sites. Becausethe selectivity and activity of proteases vary, such a library ofproteases would enable the purification of an increased number ofprotein targets.

Applicants have discovered a range of domains exhibitingtemperature-triggered self-assembly. In principle, any of these can beused in place of the tsG tag demonstrated here.

The use of centrifugation to separate assemblies is not essential. Theprinciple is separation by size, which can also be achieved byfiltration.

Example 3 Genetically-Encoded Affinity Resin (Gear) for Purification ofC-Tag Myosin V

Applicants have developed a system in which the Gus1N self-assemblingpolypeptide is genetically fused to the PDZ domain, which binds a shortpolypeptide called a C-tag. The PDZ/C-tag system has been previouslydescribed (Huang et al. 2009). Using this system, Applicants havepurified myosin V in its active form. Myosin V is a molecular motorprotein that is responsible for intracellular cargo transportation incells. The protein is a dimer which possesses so-called “legs” thatallow it to “walk” along the actin filaments, and cargo-binding domainsthat bind what the myosin actually transports, for example vesiclescontaining RNA. The process of “walking” is driven by ATP hydrolysis.Myosin V (MV) itself is a massive protein, around 137 kDa. Thesefeatures make myosin V a very difficult protein to both purify and workon. Usually, it is purified via FLAG resin, so it binds to agarose beadscoated with anti-FLAG tag antibodies and then eluted with a FLAG peptidethat outcompetes bound myosins. The anti-FLAG resin is very expensive,and just 10 mL of an anti-FLAG resin can cost about $1790.

Expression of Gus1PDZ (GEAR)

Gus1PDZ expresses very well under standard conditions, namely OD=0.6, 1mM IPTG at 30° C. and satisfactory expression is achieved after about 4hours (FIG. 16A). One advantage of the Gus1NPDZ is that only a one-steppurification is required to achieve a highly purified product. In thisexample, Gus1NPDZ was purified using affinity chromatography with theNi²⁺ column. The average yield from two separate purifications is 17.53mg/L of cell culture (FIG. 16B).

To make this process less expensive and totally lab made, Applicantscreated a fusion protein that would have a self-assembling domain as aresin-forming domain and an affinity domain having affinity to taggedMyo V. Gus1N was chosen as the resin-forming domain. Gus1N is anN-terminal domain of glutamyl-tRNA synthetase which causes either thisenzyme or different proteins tagged with it to self-assemble upon a fewminutes of heat shock. Once assembled, Gus1N-tagged molecules formcomplex meshworks. So now once Gus1N were linked to an affinity domainand heat-shocked it would provide lots of binding sites for the target.The domain we decided to choose to be fused to Gus1N to make GEAR(Genetically-Encoded Affinity Resin) is PDZ. The PDZ domain is a commonstructural domain of 80-90 amino-acids found in the signaling proteinsof bacteria, yeast, plants, viruses, and animals. PDZ is an acronymcombining the first letters of three proteins—post synaptic densityprotein (PSD95), Drosophila disc large tumor suppressor (Dlg1), andzonula occludens-1 protein (zo-1) which were first discovered to sharethe domain. PDZ domains have previously been referred to as DHR (Dlghomologous region) or GLGF (glycine-leucine-glycine-phenylalanine)domains. Engineering of its ligands allowed for the creation of a C-tag(SEQ ID NO:13). The major advantage of c-tag is that it's bound by PDZquite tightly yet is easily releasable. And this release is possible byusing the elution peptide (SEQ ID NO:14) which outcompetes proteinsbound to PDZ tagged with c-tag.

The procedure for purification is depicted in FIG. 15. Briefly, theGus1N-PDZ protein is expressed and purified from E. coli, thenheat-shocked at 48° C. for 10 minutes to cause self-assembly, generatinga solid support decorated with PDZ domains. This can be referred to as aresin. MV-Ctag is expressed in cells, which are lysed. The lysate isincubated with the resin, then centrifuged at 12,000 g for 5 minutes.The supernatant is discarded and the pellet is washed. Then elutionpeptide is added. This peptide has higher affinity for PDZ than doesCtag, so the bound MV-Ctag is released from the resin. Another 12,000 gspin is performed, and the supernatant is retained. The supernatantcontains highly purified MV-Ctag. In this purification method, thetarget protein (MV; i.e. second target protein) is never exposed to aheat shock.

Expression of Gus1PDZ

Gus1PDZ expresses very well under standard conditions, namely OD=0.6, 1mM IPTG at 30 C and satisfactory expression is achieved after about 4hours. One advantage of the Gus1NPDZ is that only one-step purificationis required to get a highly purified product. The Ni column was used forpurification of Gus1NPDZ. The average yield from two separatepurifications is 17.53 mg/L of cell culture (FIG. 16B).

To make this process less expensive and totally lab made, Applicantscreated a fusion protein that would have a self-assembling domain as aresin-forming domain and an affinity domain having affinity to taggedMyosin V. Gus1N was chosen as the resin-forming domain. Once assembled,Gus1N-tagged molecules form complex meshworks. So now once Gus1N waslinked to an affinity domain and heat-shocked it would provide a lot ofbinding sites for the target. Applicants fused PDZ to Gus1N to make GEAR(Genetically-Encoded Affinity Resin). The PDZ domain is a commonstructural domain of 80-90 amino-acids found in signaling proteins ofbacteria, yeast, plants, viruses, and animals. PDZ is an acronymcombining the first letters of three proteins—post-synaptic densityprotein (PSD95), Drosophila disc large tumor suppressor (Dlg1), andzonula occludens-1 protein (zo-1) which were first discovered to sharethe domain. PDZ domains have previously been referred to as DHR (Dlghomologous region) or GLGF (glycine-leucine-glycine-phenylalanine)domains. Engineering of the PDZ domain's natural ligand allowed for thecreation of a C-tag (SEQ ID NO:13). An advantage of C-tag is that thebinding to PDZ, though quite tightly, is easily releasable. This releaseis possible by using the elution peptide (SEQ ID NO:14) whichoutcompetes C-tagged proteins bound to PDZ.

The procedure for purification in one embodiment is depicted in FIG. 15.Briefly, the Gus1N-PDZ protein is expressed and purified from E. coli,using standard methods or FENEX. The purified Gus1N-PDZ is thenheat-shocked at 48° C. for 10 minutes to cause self-assembly, generatinga solid support decorated with PDZ domains. This can be referred to as aresin. MV-C-tag is expressed in cells, which are then lysed. The lysateis incubated with the resin, then centrifuged at 12,000 g for 5 minutes.The supernatant is discarded and the pellet is washed in bufferedsolution. Then the elution peptide is added. This peptide has a higheraffinity for the PDZ domain than the C-tag, so the bound MV-Ctag isreleased from the resin. Another 12,000 g spin is performed, and thesupernatant is retained. The supernatant contains highly purifiedMV-C-tag. In this purification method, the target protein (MV; i.e., asecond target protein) is never exposed to a heat shock.

Purification of Myosin V Using GEAR Compared to FLAG PurificationMethods

Next, GEAR purification was compared to anti-FLAG resin purification. Ascan be seen by comparing the elution lanes, MV was successfully purifiedusing GEAR in an amount that was comparable to the anti-FLAG resin. Theamount of MV purified by the Gus1NPDZ method was 25% less, however, thedegree of purity was higher, and the capacity of the resin was higher,since a large portion of uneluted MV can be seen in the resin fractioncompared to the resin fraction using FLAG purification (FIG. 20).

To test whether the purified MV was a functional protein, a glidingfilament assay was performed. In this assay, a coverslip was coated withthe GEAR-purified myosins so that the legs are exposed outward, andactin filaments that interact with the myosins were added. Next,ATP-containing buffer that triggers myosin activity was added, causingthe actin filaments to glide along the immobilized myosins. Most of theactin filaments moved, indicating that the purified myosins werefunctional.

Purification of Clover using GEAR

The binding specificity of Gus1N-PDZ was tested in two examples. In thefirst example, heat-shocked Gus1N-PDZ was incubated with eitherClover-C-tag (Clover-C) or tagless Clover. The mixture was pelleted bycentrifugation, the pellets were washed, and then visualized with bothUV and GFP channels. As shown in FIG. 17, the only pellet to emit anyfluorescence is the one incubated with C-tagged Clover. In the secondspecificity example, a similar experimental procedure is performed, butelution was achieved using the elution peptide. In FIG. 18, each samplewas split into three fractions and analyzed by SDS-PAGE: (1) resin afterelution, (2) supernatant or supe (the fraction not bound to Gus1NPDZ)and (3) eluted fractions (elution). In the case of tagless Clover,Clover protein was found in the supernatant fraction and nothing waseluted. C-tagged Clover yielded Clover-C in the elution fractions.Purified components diluted 1:10 were also loaded onto the gel as acontrol.

These results are consistent with previous results described hereinusing this method.

It was then determined whether Gus1N-PDZ could be used to purify out atarget protein from a complex lysate. To do so, Clover-C was expressedin bacteria, the lysate was incubated with pre-heat shocked Gus1NPDZ.Next, the elution was performed (FIG. 19). Clover-C was successfullypurified with only a little residual Gus1N-PDZ.

This disclosure relates to variations of the above-describedpurification method. In one variation, the target protein X is expressedas an X[cleavage site]Ctag, where [cleavage site] represents therecognition amino acid sequence for a protease, such as TEV. Instead ofan excess of elution peptide (EP), a protease-C-tag fusion protein isadded at low concentrations. The target protein is released by cleavageoff of the resin, and the protease is recruited to the resin. In furthervariations, other affinity domains and other release peptides are used.The resin concept can be used in virtually any application where beadsor other solid supports are now used, such as depletion of a targetprotein from a mixture. In virtually all cases, the fact that beads arespherical or separate from one another is irrelevant.

Although certain embodiments have been described above with a certaindegree of particularity, or with reference to one or more individualembodiments, those skilled in the art could make numerous alterations tothe disclosed embodiments without departing from the scope of thisinvention. As such, the illustrative embodiments are not intended to belimited to the particular forms disclosed. Rather, they include allmodifications and alternatives falling within the scope of the claims,and embodiments other than those shown may include some or all of thefeatures of the depicted embodiment. Further, where appropriate, aspectsof any of the examples described above may be combined with aspects ofany of the other examples described to form further examples havingcomparable or different properties and addressing the same or differentproblems. Similarly, it will be understood that the benefits andadvantages described above may relate to one embodiment or may relate toseveral embodiments.

The claims are not to be interpreted as including means-plus- orstep-plus-function limitations, unless such a limitation is explicitlyrecited in a given claim using the phrase(s) “means for” or “step for,”respectively.

REFERENCES

The following references (including patent documents and non-patentliterature), to the extent that they provide exemplary procedural orother details supplementary to those set forth herein, are eachspecifically incorporated herein by reference, each in its entirety.

-   Nover, L., Scharf, K. D. & Neumann, D. Formation of cytoplasmic heat    shock granules in tomato cell cultures and leaves. Molecular and    cellular biology 3, 1648-1655 (1983).-   Grousl, T. et al. Robust heat shock induces    eIF2alpha-phosphorylation-independent assembly of stress granules    containing eIF3 and 40S ribosomal subunits in budding yeast,-   Saccharomyces cerevisiae. Journal of cell science 122, 2078-2088,    doi:10.1242/jcs.045104 (2009).-   Grousl, T. et al. Heat shock-induced accumulation of translation    elongation and termination factors precedes assembly of stress    granules in S. cerevisiae. PloS one 8, e57083,    doi:10.1371/journal.pone.0057083 (2013).-   Rinnerthaler, M. et al. Mmil, the yeast homologue of Mammalian TCTP,    associates with stress granules in heat-shocked cells and modulates    proteasome activity. PloS one 8, e77791,    doi:10.1371/journal.pone.0077791 (2013).-   Kimball, S. R., Horetsky, R. L., Ron, D., Jefferson, L. S. &    Harding, H. P. Mammalian stress granules represent sites of    accumulation of stalled translation initiation complexes. American    journal of physiology. Cell physiology 284, C273-284,    doi:10.1152/ajpcell.00314.2002 (2003).-   Kedersha, N. & Anderson, P. Regulation of translation by stress    granules and processing bodies. Progress in molecular biology and    translational science 90, 155-185, doi:10.1016/S1877-1173(09)90004-7    (2009).-   Anderson, P. & Kedersha, N. Stress granules: the Tao of RNA triage.    Trends in biochemical sciences 33, 141-150,    doi:10.1016/j.tibs.2007.12.003 (2008).-   Yao, G. et al. PAB1 self-association precludes its binding to    poly(A), thereby accelerating CCR4 deadenylation in vivo. Molecular    and cellular biology 27, 6243-6253, doi:10.1128/MCB.00734-07 (2007).-   Simon, E. & Seraphin, B. A specific role for the C-terminal region    of the Poly(A)-binding protein in mRNA decay. Nucleic acids research    35, 6017-6028, doi:10.1093/nar/gkm452 (2007).-   Simader, H. et al. Structural basis of yeast aminoacyl-tRNA    synthetase complex formation revealed by crystal structures of two    binary sub-complexes. Nucleic acids research 34, 3968-3979,    doi:10.1093/nar/gk1560 (2006).-   Simader, H., Hothorn, M. & Suck, D. Structures of the interacting    domains from yeast glutamyl-tRNA synthetase and tRNA-aminoacylation    and nuclear-export cofactor Arc1p reveal a novel function for an old    fold. Acta crystallographica. Section D, Biological crystallography    62, 1510-1519, doi:10.1107/S0907444906039850 (2006).-   Graindorge, J. S., Scngcr, B., Tritch, D., Simos, G. & Fasiolo, F.    Role of Arc1p in the modulation of yeast glutamyl-tRNA synthetase    activity. Biochemistry 44, 1344-1352, doi:10.1021/bi049024z (2005).-   Ananthan, J., Goldberg, A. L. & Voellmy, R. Abnormal proteins serve    as eukaryotic stress signals and trigger the activation of heat    shock genes. Science 232, 522-524 (1986).-   Trotter, E. W. et al. Misfolded proteins are competent to mediate a    subset of the responses to heat shock in Saccharomyces cerevisiae.    The Journal of biological chemistry 277, 44817-44825 (2002).-   Mitchell, S. F., Jain, S., She, M. & Parker, R. Global analysis of    yeast mRNPs. Nature structural & molecular biology 20, 127-133,    doi:10.1038/nsmb.2468 (2013).-   Price-Carter, M., Fazzio, T. G., Vallbona, E. I. & Roth, J. R.    Polyphosphate kinase protects Salmonella enterica from weak organic    acid stress. Journal of bacteriology 187, 3088-3099,    doi:10.1128/JB.187.9.3088-3099.2005 (2005).-   Anderson, P. & Kedersha, N. Stressful initiations. Journal of cell    science 115, 3227-3234 (2002).-   Barany & Merrifield, In: The Peptides, Gross and Meienhofer (Eds.),    Academic Press, NY, 1-284, 1979 (1979)-   Huang et al., J. Mol. Biol. 392:1221-21 (2009)-   Lee et al., PLoS One 8:367902 (2013)-   Merrifield, Science, 232(4748):341-347, (1986)-   Sha et al., PNAS 110:14924-9 (2013)-   Stewart & Young, In: Solid Phase Peptide Synthesis, 2d. ed., Pierce    Chemical Co., 1984-   Tam et al., J. Am. Chem. Soc., 105:6442, 1983 (1983)-   Netzger et al. (2009), “Innate immune and chemically triggered    oxidative stress modifies translational fidelity,” Nature    462:522-526-   U.S. Pat. No. 5,206,347

1. A self-assembling fusion protein comprising: (a) a heat-inducibleself-assembly domain; and (b) a target protein; wherein theself-assembly domain remains folded during assembly.
 2. (canceled) 3.The fusion protein of claim 1, wherein the self-assembly domain is aGST-like domain or a polypeptide with at least 90% identity to aGST-like domain.
 4. The fusion protein of claim 1, wherein theself-assembly domain comprises a polypeptide from Arc1, Mes1, Gus1, or apolypeptide with at least 90% identity to Arc1, Mes1, or Gus1.
 5. Thefusion protein of claim 4, wherein the self-assembly domain comprises apolypeptide that is at least 20 amino acids in length and has at least90% identity to 20 contiguous amino acids of the first 250 amino acidsfrom Arc1, Mes1, or Gus1.
 6. The fusion protein of claim 4, wherein theself-assembly domain comprises a polypeptide from Gus1 or a polypeptidewith at least 90% identity to Gus1 or a fragment thereof.
 7. The fusionprotein of claim 1, wherein the self-assembly domain comprises apolypeptide from Tef3, Tef4, Efb1, or a polypeptide with at least 90%identity to Tef3, Tef4, Efb1, or fragments thereof.
 8. (canceled)
 9. Thefusion protein of claim 1, wherein the self-assembly domain comprises apolypeptide from Yef3, Ura7, or a polypeptide with at least 90% identityto Yef3 Ura7, or fragments thereof.
 10. (canceled)
 11. The fusionprotein of claim 1, wherein the self-assembly domain is a polypeptidefrom a Saccharomyces cerevisiae protein or a polypeptide with at least90% identity to a Saccharomyces cerevisiae protein.
 12. The fusionprotein of claim 1, wherein the self-assembly domain comprises apolypeptide of SEQ ID NO:9, 10, 11, or 12, a fragment thereof, or apolypeptide with at least 90% identity to SEQ ID NO:9, 10, 11, or 12, ora fragment thereof. 13-14. (canceled)
 15. The fusion protein of claim 1,wherein the self-assembly domain is between 20 and 250 amino acids inlength.
 16. The fusion protein of claim 1, wherein the target protein isferritin, a fluorescent protein, an antibody, an antibody fragment,protein A, streptavidin, protein G, protein A/G, protein L, a protease,PDZ domain or StrepTactin.
 17. The fusion protein of claim 1, whereinthe fusion protein further comprises a protease cleavage site betweenthe target protein and the self-assembly domain. 18-20. (canceled)
 21. Aprotein aggregate comprising the fusion protein of claim
 1. 22-23.(canceled)
 24. A polynucleotide encoding for the fusion protein ofclaim
 1. 25. A host cell comprising the fusion protein of claim
 1. 26.Cell lysate comprising the fusion protein of claim
 1. 27. A method foraggregating a target protein comprising: formulating an aqueouscomposition comprising the fusion protein of claim 1; and heating thecomposition to a temperature between about 35 and 50° C. 28-30.(canceled)
 31. A method of immunoprecipitating or purifying a moleculecomprising the steps of: formulating a first composition comprising thefusion protein of claim 1; wherein the target protein is a first targetprotein that specifically binds to the molecule; heating the firstcomposition to a temperature between about 35 and 50° C. to form proteinaggregates comprising the fusion protein; and contacting the firstcomposition with a second composition comprising the molecule. 32-45.(canceled)
 46. A method for purifying a protein comprising: formulatingan aqueous composition comprising a fusion protein of claim 1; heatingthe aqueous composition to a temperature between about 35 and 50° C. toform protein aggregates comprising the fusion protein; separating theprotein aggregates from the aqueous composition. 47-49. (canceled) 50.The fusion protein of claim 1, wherein the target protein is arestriction enzyme, DNA polymerase, protease, ligase, RNA polymerase,methylase, polyadenylate polymerase, topoisomerase, guanylyltransferase, ribonuclease, deoxyribonuclease, alkaline phosphatase,polynucleotide kinase or reverse transcriptase.